Identification of Islet Proteins Stimulatory for T cells from Diabetes Patients. Abstract. Type 1 Diabetes Mellitus (T1DM) is a cell-mediated autoimmune disease directed against the beta cells whereas Type 2 diabetes (T2DM) is not autoimmune. In recent years, a group of autoimmune phenotypic T2DM patients have been described suggesting that T cell mediated autoimmunity may be associated with the pathogenesis and progression of beta cell failure in both T1DM and T2DM. In two masked NIH sponsored workshops, our cellular immunoblotting T cell assay, which uses isolated human islets, has been validated to distinguish T1DM patients from controls with excellent specificity and sensitivity. Interestingly, T cells from T1DM patients respond to select molecular weight regions of islet proteins (preliminary data) as identified using cellular immunoblotting. Therefore, we hypothesize that individual islet proteins are responsible for the T cell reactivity observed in autoimmune diabetes patients and that identifying these proteins will provide insight into T cell reactivity in autoimmune diabetes. Previously, islet proteins utilized in T cell assays were identified based on autoantibody reactivity and not direct T cell reactivity. What is currently needed is the identification of islet proteins that directly stimulate T cells from autoimmune diabetes patients. In collaboration with experts in proteomics, we propose to isolate and identify islet proteins stimulatory to T cells from autoimmune diabetes patients using the following three approaches: a). Isolation of T cell stimulatory proteins from molecular weight regions of interest, identified by cellular immunoblotting, using electro-elution of one and two-dimensional SDS-PAGE followed by tandem mass spectrometric (MS/MS) identification of proteins. b). Sequential biochemical approaches to isolate proteins to near homogeneity followed by MS/MS identification of the purified proteins. c). A novel orthogonal separation strategy to identify proteins that activate T cells. Once the T cell stimulatory islet proteins are isolated and identified, we will proceed to verify the isolated islet proteins by analysis of T cell reactivity in autoimmune diabetes patients. Identification of individual antigenic islet cell proteins that directly stimulate T cells from diabetes patients could lead to the development of new antigen based assays and intervention strategies, and provide innovative methods to follow and assess new therapeutic trials. PUBLIC HEALTH RELEVANCE: Autoimmune diseases affect 5-7% of the adult population in North America and Europe. We propose to identify and isolate the islet proteins which stimulate the autoreactive T cells in patients who develop autoimmune diabetes (type 1 diabetes and a subset of type 2 diabetes). The results of these studies will have major implications toward furthering our understanding of the development of autoimmune disease. Subsequent identification of potential islet proteins will be useful in the development of new antigen based intervention strategies and new assays to monitor immunomodulatory therapies for treating autoimmune diabetes.